Exposure of Culex quinquefasciatus to the oomycete Pythium insidiosum: A protocol for in vitro studies

Pythium insidiosum causes pythiosis, an infection that affects different species of mammals, including humans, and inhabits marshy ecosystems of tropical, subtropical, and temperate regions worldwide. Therefore, this study proposes a protocol to expose Culex quinquefasciatus to P. insidiosum zoospores. Cx. quinquefasciatus immatures (eggs, larvae, and pupae) were exposed to zoospores (8x103 zoospores/mL) of the oomycete for 24 h. The exposure of Cx. quinquefasciatus to the zoospores from L1 to the emergence of adults was evaluated, and P. insidiosum detection was performed by microbiological culture, poly-merase chain reaction, and histopathological analysis of stage 4 larvae. The protocol used to produce Cx. quinquefasciatus colonies and adapted for this study proved viable for research on the interaction be-tween P. insidiosum and this Culicidae species. Moreover, P. insidiosum presence was evident in all larval stages of the mosquito, although the presence of the oomycete was not detected in the eggs, pupae, and adults. This study is a pioneer in the development of a protocol to evaluate Cx. quinquefasciatus exposure to P. insidiosum zoospores, and under experimental conditions, P. insidiosum can establish itself in Cx. quinquefasciatus larval stages. The developed protocol is expected to serve as a basis for developing studies to evaluate the interactions of P. insidiosum with these mosquitoes and shed more light on the participation of culicids in expanding the ecological niche of P. insidiosum.(c) 2023 British Mycological Society. Published by Elsevier Ltd. All rights reserved.